Osteoclasts (OCLs) are the only bone-resorbing cells that are essential for normal skeletal development and bone remodeling throughout life. Abnormal OCL number and/or activity result in a number of bone diseases such as osteoporosis, Paget's disease of bone, metastatic osteolytic lesions, and rheumatoid arthritis. However, the molecular mechanisms underlying osteoclastogenesis are not well understood. Our preliminary studies demonstrate that activating transcription factor 4 (ATF4) is a key factor that regulates osteoclastogenesis. Our data indicate that ATF4 mediates M-CSF-induced expression of RANK, a critical molecular event required for early differentiation of OCLs. Furthermore, ATF4 directs RANKL-dependent gene expression of NFATc1, a master regulator of OCL differentiation. In this study, we hypothesize that ATF4 plays a critical role in regulating osteoclastogenesis by two distinct mechanisms: 1) ATF4 modulates M-CSF induction of RANK expression via PI3K/AKT-dependent phosphorylation and protein stabilization and/or activation;2) ATF4 mediates RANKL induction of NFATc1 gene by binding to the NFATc1 gene P1 promoter via interactions with other key factors. To address our hypothesis, we will pursue the following specific aims: Aim 1 will confirm that ATF4 is phosphorylated and up-regulated by M-CSF signaling via PI3K/AKT in OCL precursors. We will identify the AKT and M-CSF responsive phosphorylation site(s) and assess their functional significance in regulating ATF4 protein stability and activity in support of osteoclastogenesis. Aim 2 will determine whether ATF4 mediates RANKL induction of the NFATc1 gene via cooperative interaction with other key factors on the NFATc1 gene P1 promoter. We will also identify ATF4 heterodimerization partners and assess the functional significance of interactions between ATF4 and partners in RANKL-induced NFATc1 expression. Aim 3 will determine whether OCL-specific transgenic expression of NFATc1 can rescue the defect in OCL differentiation in Atf4-/- mice. We will use transgenic mice in which the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter drives the expression of a constitutively active form of NFATc1 (NFATc1-CA) selectively in OCLs in Atf4-/- mice. Biochemical and histomorphometric parameters for OCL differentiation and bone resorption will be determined in the following mice groups: i) wt, ii) NFATc1- CA-tg, iii) Atf4-/-, and iv) Atf4-/-;NFATc1-CA-tg. Successful completion of these proposed aims will 1) significantly advance understanding of the molecular mechanisms underlying OCL differentiation, and 2) provide a molecular basis for development of new and more specific antiresorptive agents for treating patients with devastating osteoporosis, hypercalcemia of malignancy, and Paget's disease of bone. PUBLIC HEALTH RELEVANCE: Skeletal integrity requires a delicate balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCLs). Abnormally increased OCL number and/or activity result in a number of bone diseases such as osteoporosis, osteolytic lesions induced by many metastatic cancers, Paget's disease of bone, and rheumatoid arthritis. Conversely, reduced OCL number and/or activity causes osteopetrosis, a disorder characterized by significantly increased skeletal mass. Defining the molecular mechanisms underlying osteoclastogenesis is essential to advance understanding of the molecular basis for the pathogenesis of OCL-based or involved bone diseases and improve the prevention and treatment of these diseases. We demonstrate that ATF4 is a key transcription factor for osteoclastogenesis and present data revealing its importance in the regulation of both early and late OCL differentiation. ATF4 plays an intrinsic role in OCL precursors that is indispensable for RANKL-induced OCL differentiation. The important role of ATF4 in osteoclastogenesis is underscored by its requirements for M-CSF-induced RANK gene expression, a key molecular event for early OCL differentiation, as well as for RANKL-induced NFATc1 gene expression, required for OCL differentiation. This proposal intends to elucidate the mechanisms whereby M-CSF activates/upregulates ATF4 via the PI3K/AKT pathway, to examine how ATF4 mediates RANKL induction of NFATc1 via activation of the NFATc1 gene P1 promoter, and to assess the importance of ATF4 in OCL differentiation in vivo using a specific NFATc1-CA transgenic mouse model. The information obtained from these studies will significantly enhance our understanding of the molecular mechanism involved in normal osteoclastogenesis and bone resorption during skeletal development and throughout life. Furthermore, bisphosphonates (zolendronic acid and pamidronate), the most widely prescribed antiresorptive agents that are intravenously administrated to reduce bone pain, hypercalcemia and skeletal complications in patients with multiple myeloma, breast, prostate, lung and other cancers and Paget's disease of bone, have a severe side effect called bisphosphonate- associated osteonecrosis of the jaw via undefined mechanism(s). Successful completion of this study will provide a molecular basis for the development of new and more specific antiresorptive agents for treating these devastating diseases.